We reviewed the records of 421 singletons naturally born and 482 singletons born by IVF-ET in our hospital during 2013–2016. We evaluated the fetal growth measurement and ratio of umbilical cord end systolic peak over end diastolic peak (S/D). Furthermore, we recruited 21 singletons born by fresh IVF-ET and 22 singletons born by naturally conceived (NC) from Jan.1, 2016- Jan.1, 2017 for evaluation of the function of human umbilical endothelial cells. Baselines of parental characteristics were collected in the third trimester; these included maternal blood pressure, heart rate, the serum levels of fasting blood glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, homocysteine, and serum estradiol concentration.
Children born with cardiovascular diseases, congenital anomalies, premature delivery, small for gestational age were excluded. We also excluded children whose mothers had gestational complications, such as gestational diabetes, preeclampsia, and others.
Umbilical vessels and cord blood were collected from twenty-one IVF and twenty-two NC singleton pregnancies immediately after cesarean delivery in the Women’s Hospital. The detailed characteristics of these samples are listed in Supplementary Table 1. Mothers with previous cardiovascular diseases, or other gestational complications were also excluded. Mothers of IVF babies had normal ovarian function and experienced controlled ovarian hyper-stimulation with gonadotropins followed by the standard luteal long gonadotropin-releasing hormone agonist down-regulation protocol for the first IVF cycle. Embryo transfer was performed after 2-3 days of egg retrieval. All babies included met the following criteria: maternal age between 25 and 35 years old; full-term delivery; singleton pregnancy; child’s birth weight between 2500-4000 g; no indication of pregnancy complication; no birth defects; and no cardiovascular diseases. We examined the E2 levels in cord blood from newborns with the E2 kit (H-Estradiol E2, Abcam, ab108640).
Isolation and culture of Primary human umbilical Vein Endothelial Cells
The protocols for HUVEC isolation and culture were performed and modified according to Crampton et. al.. Briefly, the fresh vein was filled with a solution containing 1mg/ml collagenase, the cord was incubated in pre-warmed phosphate-buffered saline at 37 ℃ for 30 minutes, and cells were cultured in 5% fetal bovine serum combined with endothelial cell medium (ECM, ScienCell, cat. #1001). The HUVECs were used between passage 3 to 5.
Small interfering RNA-mediated MEG3 knock-down
SiRNA oligonucleotides were purchased from Thermo Fisher Scientific. The sequences of siRNAs targeting Meg3 are as follows (5′–3′): sense, GCUCAUACUUUGACUCUAUTT; anti-sense, AUAGAGUCAAAGUAUGAGCTT. The sequences of negative control (NC) are as follows (5′–3′): sense, UUCUCCGAACGUGUCACGUdTdT; anti-sense, ACGUGACACGUUCGGAGAAdTdT. The siRNAs against Meg3 were reverse-transfected into cells at a dose of 10nm in 6-wells plates using the Lipofectamine 3000 reagent (ThermoFisher, Catalog. L3000008) for 48h.
Quantitative Realtime PCR Analysis
Total RNA was extracted from the tissue sample and primary human umbilical vessel cells using the TRIzol Reagents (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was synthesized using PrimerScript RT Reagent Kit (Takara, RR037A, Japan) in a 20 µl reaction containing 0.5-1ug of total RNA. Realtime quantitative PCR was performed using ABI Prism 7900HT (Applied Biosystem, Foster City, CA). GAPDH was the internal control. Full list of primer sequences is shown in Supplementary Table 2.
The protein was extracted from HUVECs tissues with lysis buffer, which was separated using 10% SDS-PAGE. Western Blots was performed using polyvinylidene fluoride membrane and the antibodies for DNMT3A (Cell Signaling, 32578, used at a dilution of 1:1000), DNMT3B (Cell Signaling, 57868, used at a dilution of 1:1000), beta-ACTIN (Abcam, ab8226, used at a dilution of 1:5000). Protein bands were visualized by the enhanced chemiluminescence system (Pierce, Rockford, IL).
Umbilical cord blood and cell supernatants were collected after cesarean delivery. ELISA kits were used for determination of NO (Nitric Oxide Assay, ab65328, abcam, UK), VEGF (H-VEGF, DVE00, R&D, USA), and ET-1 (H-Endothelin-1, QET00B, R&D, USA) levels. The procedures were performed according to the manufacturers' protocols. For the cell-line culture, three replicates of data were used for statistical analyses.
DNA isolation and bisulfite conversion
Total genomic DNA was isolated from umbilical vessel tissues using Genomic DNA Purification Kit (Invitrogen, cat. K0512, USA). Bisulfite was converted using the EpiTect bisulfite kit (Qiagen, Valencia, CA) according to the manufacturerer’s instructions to deaminate cytosine to uracil; 5-methyl-cytosine was protected from deamination. PCRs were performed in a ABI 9700 PCR System (Applied Biosystems, USA) using an annealing temperature of 56°C.
DNA Methylation analysis by pyrosequencing
The bisulfite converted DNA was amplified using Hotstart Plus DNA polymerase (Qiagen). PCR products were immobilized on streptavidin-sepharose beads (GE Healthcare), washed, denatured and released into annealing buffer containing sequencing primer which is described in Supplementary Table 3. Pyrosequencing was carried out on a PyroMark Q96 instrument (Qiagen) according to the manufacturer's instruction. % methylation was calculated using the Pyro Q CpG software (Qiagen).
Data were analyzed using SPSS 18.0, and were presented as mean ± SD or mean ± SE. Statistical analysis including unpaired two-tailed Student’s t-test was performed as described in the figure legends or Excel legends. P < 0.05, P<0.01, or P<0.001 was considered statistically significant.